Increased toxicity by transforming growth factor-beta 1 in liver cells overexpressing CYP2E1

Ethanol treatment causes an increase in expression of TGF-beta1 and CYP2E1 in the centrilobular area. Alcoholic liver disease is usually initiated in the centrilobular region of the liver. We hypothesized that the combination of TGF-beta1 and CYP2E1 produces increased oxidative stress and liver cell toxicity. To test this possibility, we studied the effects of TGF-beta1 on the viability of HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells, which do not express CYP2E1. E47 cells underwent greater growth inhibition and enhanced apoptosis after TGF-beta1 treatment, as compared to the C34 cells. There was an enhanced production of reactive oxygen species (ROS) and a decline in reduced glutathione (GSH) levels in the TGF-beta1-treated E47 cells and the enhanced cell death could be prevented by antioxidants. The CYP2E1 inhibitor diallyl sulfide prevented the potentiated cell death in E47 cells validating the role of CYP2E1. Mitochondrial membrane potential declined in the TGF-beta1-treated E47 cells, prior to developing toxicity, and cell death could be prevented by trifluoperazine, an inhibitor of the mitochondrial membrane permeability transition. TGF-beta1 also produced a loss of cell viability in hepatocytes from pyrazole-treated rats with elevated levels of CYP2E1, compared to control hepatocytes. In conclusion, increased toxic interactions by TGF-beta1 plus CYP2E1 can occur by a mechanism involving increased production of intracellular ROS and depletion of GSH, resulting in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of TGF-beta1-induced cell death by CYP2E1 may contribute to mechanisms of alcohol-induced liver disease.     

Serum deprivation-induced HepG2 cell death is potentiated by CYP2E1

Induction of oxidative stress plays a key role in serum deprivation-induced apoptosis. CYP2E1 plays an important role in toxicity of many chemicals and ethanol and produces oxidant stress. We investigated whether CYP2E1 expression can sensitize HepG2 cells to toxicity as a consequence of serum deprivation. The models used were HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells which do not express CYP2E1. E47 cells showed greater growth inhibition and enhanced cell death after serum deprivation, as compared to the C34 cells. DNA ladder and flow cytometry assays indicated that apoptosis occurred at earlier times after serum deprivation in E47 than C34 cells. Serum withdrawal-induced E47 cell death could be rescued by antioxidants, the mitochondrial permeability transition inhibitor cyclosporine A, z-DEVD-fmk, and a CYP2E1 inhibitor 4-methylpyrazole. Increased production of reactive oxygen species (ROS) and lipid peroxidation occurred in E47 cells after serum deprivation, and there was a corresponding decline in the E47 cell mitochondrial membrane potential and reduced glutathione (GSH) levels. We propose that the mechanism of this serum withdrawal plus CYP2E1 toxicity involves increased production of intracellular ROS, lipid peroxidation, and decline of GSH levels, which results in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of serum deprivation-induced cell death by CYP2E1 may contribute to the sensitivity of the liver to alcohol-induced ischemia and growth factor deprivation.     

Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells

Thioredoxin is an important reducing molecule in biological systems. Increasing CYP2E1 activity induces oxidative stress and cell toxicity. However, whether thioredoxin protects cells against CYP2E1-induced oxidative stress and toxicity is unknown. SiRNA were used to knockdown either cytosolic (TRX-1) or mitochondrial thioredoxin (TRX-2) in HepG2 cells expressing CYP2E1 (E47 cells) or without expressing CYP2E1 (C34 cells). Cell viability decreased 40-60% in E47 but not C34 cells with 80-90% knockdown of either TRX-1 or TRX-2. Depletion of either thioredoxin also potentiated the toxicity produced either by a glutathione synthesis inhibitor or by TNFα in E47 cells. Generation of reactive oxygen species and 4-HNE protein adducts increased in E47 but not C34 cells with either thioredoxin knockdown. GSH was decreased and adding GSH completely blocked E47 cell death induced by either thioredoxin knockdown. Lowering TRX-1 or TRX-2 in E47 cells caused an early activation of ASK-1, followed by phosphorylation of JNK1 after 48 h of siRNA treatment. A JNK inhibitor caused a partial recovery of E47 cell viability after thioredoxin knockdown. In conclusion, knockdown of TRX-1 or TRX-2 sensitizes cells to CYP2E1-induced oxidant stress partially via ASK-1 and JNK1 signaling pathways. Both TRX-1 and TRX-2 are important for defense against CYP2E1-induced oxidative stress.     

Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1

CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells.     

The CYP2E1 inhibitor DDC up-regulates MMP-1 expression in hepatic stellate cells via an ERK1/2- and Akt-dependent mechanism

DDC (diethyldithiocarbamate) could block collagen synthesis in HSC (hepatic stellate cells) through the inhibition of ROS (reactive oxygen species) derived from hepatocyte CYP2E1 (cytochrome P450 2E1). However, the effect of DDC on MMP-1 (matrix metalloproteinase-1), which is the main collagen degrading matrix metalloproteinase, has not been reported. In co-culture experiments, we found that DDC significantly enhanced MMP-1 expression in human HSC (LX-2) that were cultured with hepatocyte C3A cells either expressing or not expressing CYP2E1. The levels of both proenzyme and active MMP-1 enzyme were up-regulated in LX-2 cells, accompanied by elevated enzyme activity of MMP-1 and decreased collagen I, in both LX-2 cells and the culture medium. H₂O₂ treatment abrogated DDC-induced MMP-1 up-regulation and collagen I decrease, while catalase treatment slightly up-regulated MMP-1 expression. These data suggested that the decrease in ROS by DDC was partially responsible for the MMP-1 up-regulation. ERK1/2 (extracellular signal-regulated kinase 1/2), Akt (protein kinase B) and p38 were significantly activated by DDC. The ERK1/2 inhibitor (U0126) and Akt inhibitor (T3830) abrogated the DDC-induced MMP-1 up-regulation. In addition, a p38 inhibitor (SB203580) improved MMP-1 up-regulation through the stimulation of ERK1/2. Our data indicate that DDC significantly up-regulates the expression of MMP-1 in LX-2 cells which results in greater MMP-1 enzyme activity and decreased collagen I. The enhancement of MMP-1 expression by DDC was associated with H₂O₂ inhibition and coordinated regulation by the ERK1/2 and Akt pathways. These data provide some new insights into treatment strategies for hepatic fibrosis.     

Alcohol steatosis and cytotoxicity: The role of cytochrome P4502E1 and autophagy

The goal of the current study was to evaluate whether CYP2E1 plays a role in binge-ethanol induced steatosis and if autophagy impacts CYP2E1-mediated hepatotoxicity, oxidative stress and fatty liver formation produced by ethanol. Wild type (WT), CYP2E1 knockin (KI) and CYP2E1 knockout (KO) mice were gavaged with 3g/kg body wt ethanol twice a day for four days. This treatment caused fatty liver, elevation of CYP2E1 and oxidative stress in WT and KI mice but not KO mice. Autophagy was impaired in ethanol-treated KI mice compared to KO mice as reflected by a decline in the LC3-II/LC3-I ratio and lower total LC-3 and Beclin-1 levels coupled to increases in P62, pAKT/AKT and mTOR. Inhibition of macroautophagy by administration of 3-methyladenine enhanced the binge ethanol hepatotoxicity, steatosis and oxidant stress in CYP2E1 KI, but not CYP2E1 KO mice. Stimulation of autophagy by rapamycin blunted the elevated steatosis produced by binge ethanol. Treatment of HepG2 E47 cells which express CYP2E1 with 100mM ethanol for 8 days increased fat accumulation and oxidant stress but decreased autophagy. Ethanol had no effect on these reactions in HepG2 C34 cells which do not express CYP2E1. Inhibition of autophagy elevated ethanol toxicity, lipid accumulation and oxidant stress in the E47, but not C34 cells. The antioxidant N-acetylcysteine, and CYP2E1 inhibitor chlormethiazole blunted these effects of ethanol. These results indicate that CYP2E1 plays an important role in binge ethanol-induced fatty liver. We propose that CYP2E1-derived reactive oxygen species inhibit autophagy, which subsequently causes accumulation of lipid droplets. Inhibition of autophagy promotes binge ethanol induced hepatotoxicity, steatosis and oxidant stress via CYP2E1.     

Overexpression of CYP2E1 induces HepG2 cells death by the AMP kinase activator 5′-aminoimidazole-4-carboxamide-1-β-<Emphasis Type="SmallCaps">d</Emphasis>-ribofuranoside (AICAR)

5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a phylogenetically conserved serine/threonine protein kinase. AMPK may inhibit cell growth and proliferation and also regulates apoptosis. 5′-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) is a cell-permeable AMPK activator. Activation of AMPK with AICAR has been shown to induce apoptosis of the rat hepatoma cell line FTO2B cells and almost completely inhibited HepG2 cells growth. In this study, a HepG2 cell line, which was transfected with a vector containing human CYP2E1 cDNA (E47 cells), was treated with AICAR. Cell proliferation was blocked, and apoptosis and necrosis were elevated as assessed by cellular morphology, DNA content assay, and lactate dehydrogenase leakage. AICAR treatment significantly increases CYP2E1 activity (20-fold) and expression (5.5-fold) in E47 cells. Iodotubericidin, which inhibits the conversion of AICAR to its activated form AICAR monophosphate, the antioxidants trolox and MnTMPyP, and 4-methylpyrazole, an inhibitor of CYP2E1, all can protect the E47 cells from AICAR-induced necrosis. Production of intracellular reactive oxygen species was increased by AICAR treatment in E47 cells. The cytotoxicity mechanism of AICAR in E47 cells is suggested to include AMPK activation, p53 phosphorylation, p21 expression, overexpression of CYP2E1, and intracellular ROS accumulation.     

Geldanamycin, an inhibitor of Hsp90 increases cytochrome P450 2E1 mediated toxicity in HepG2 cells through sustained activation of the p38MAPK pathway

Cytochrome P450 2E1 (CYP2E1) can mediate reactive oxygen species (ROS) induced cell death through its catalytic processes. Heat shock protein 90 (Hsp90) is an important molecular chaperone which is essential for cellular integrity. We previously showed that inhibition of Hsp90 with Geldanamycin (GA), an inhibitor of Hsp90 increased CYP2E1 mediated toxicity in CYP2E1 over-expressing HepG2 cells (E47 cells) but not in C34-HepG2 cells devoid of CYP2E1 expression. The aim of the present study was to test the hypothesis that the potentiation of CYP2E1 toxicity in E47 cells with GA may involve changes in mitogen activated protein kinase signal transduction pathways. GA was toxic to E47 cells and SB203580, an inhibitor of p38 MAPK prevented this decrease in viability. The protective effects of SB203580 were effective only when SB203580 was added before GA treatment. GA activated p38 MAPK in E47 cells and this activation was an early and a sustained event. GA elevated ROS levels and lipid peroxidation and lowered GSH levels in E47 cells and these changes were blunted or prevented by treatment with SB203580. Apoptosis was increased by GA and prevented by pre-treatment with SB203580. The loss in mitochondrial membrane potential in E47 cells after GA treatment was also decreased significantly with SB203580 treatment. The activity and expression of CYP2E1 and Hsp90 levels were not altered by SB203580. In conclusion, the inhibition of Hsp90 with GA increases the toxicity of CYP2E1 in HepG2 cells through an early and sustained activation of the p38 MAPK pathway.     

Heterogenous induction of carcinoma-associated fibroblast-like differentiation in normal human prostatic fibroblasts by co-culturing with prostate cancer cells

In the tumor microenvironment, carcinoma-associated fibroblasts (CAFs) are considered to play a critical role in the promotion of tumorigenesis. However, the mechanisms that generate CAFs are not well elucidated. To understand how CAFs are generated during primary cancer progression, we investigated the biochemical characteristics of normal human prostate stromal cells (PrSC) co-cultured with human prostate cancer (PCa) cells in vitro. In primary cultures of human PCa-derived stromal cells (PCaSC-8 and PCaSC-9), expression of TNC, ACTA2, EGF, FGF7, and IGF1 mRNA was generally higher than PrSC but gene expression patterns were not uniform between PCaSC-8 and PCaSC-9 cells. Transforming growth factor β (TGFβ) and vascular endothelial growth factor (VEGF) protein levels in both PCaSC-8 and PCaSC-9 cells were generally higher than PrSC but levels of both secreted proteins were not same. When PrSCs were co-cultured with androgen-sensitive LNCaP cells or its sublines, androgen-low-sensitive E9 cells and androgen-insensitive AIDL cells, mRNA expression of IGF1 was significantly increased in all combinations. In contrast, expression of COL1A1, TNC, and ACTA2 mRNA was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Protein production of VEGF was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Increase of TGFβ protein was observed only in E9 + PrSC co-cultures. These biochemical characteristics of PrSC were partially recapitulated in TGFβ-treated PrSC. We have demonstrated that normal fibroblasts co-cultured with cancer cells become activated and exhibit biochemical characteristics of CAFs in a heterogenous manner. Our results suggest that heterogenous induction of CAF-like differentiation might be strongly dependent on biochemical characteristics of adjacent cancer cells.     

Estrogenic modulation of CYP3A38, CYP3A40, and CYP19 in mature male medaka (Oryzias latipes)

We examined cytochrome P450 production and activity and circulating hormone concentrations in male medaka exposed to 17beta-estradiol (E2) or 17alpha-ethinylestradiol (EE2). Intraperitoneal injection of E2 at 1, 10, or 100 microg/g-fish completely suppressed CYP3A38 protein production and suppressed CYP3A40 protein levels by 89%, 52%, or 47%, respectively. CYP3A38 and CYP3A40 mRNA expression was unaltered, and CYP3A enzymatic activity initially increased and then decreased with increasing E2 dose. Males co-cultured with females were exposed to a markedly high concentration (43 ng/L) of E2 secreted by females. CYP3A protein levels in co-cultured males were suppressed. Serum testosterone (TE) and 11keto-testosterone levels in co-cultured males were downregulated to 40% of pre-exposure levels. Serum E2 levels increased in co-cultured males or males exposed to EE2. Testicular CYP19, which converts TE to E2, increased by 9.5 times in males exposed to 50 ng/L EE2 and by 21.5 times in those exposed to 100 ng/L EE2. Male medaka exposed to EE2 showed increased serum Vtg levels. Estrogenic exposure induced Vtg production, suppressed CYP3A protein production, downregulated TE metabolism, and enhanced CYP19 activity. Serum E2 endogenously induced by CYP19 could contribute to Vtg induction in male medaka.     

Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

Introduction

The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs.

Methods

Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR.

Results

Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs.

Conclusions

Co-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.     

Impaired Endothelium-Mesenchymal Stem Cells Cross-talk in Systemic Sclerosis: a Link Between Vascular and Fibrotic Features

Introduction

To assess if an impaired cross-talk between endothelial cells (ECs) and perivascular/multipotent mesenchymal stem cells (MSCs) might induce a perturbation of vascular repair and leading to a phenotypic switch of MSC toward myofibroblast in Systemic Sclerosis (SSc).

Methods

We investigated different angiogenic and profibrotic molecules in a tridimentional matrigel assay, performing co-cultures with endothelial cells (ECs) and bone marrow derived MSCs from patients and healthy controls (HC). After 48 hours of co-culture, cells were sorted and analyzed for mRNA and protein expression.

Results

ECs-SSc showed a decreased tube formation ability which is not improved by co-cultures with different MSCs. After sorting, we showed: i. an increased production of vascular endothelial growth factor A (VEGF-A) in SSc-MSCs when co-cultured with SSc-ECs; ii. an increased level of transforming growth factor beta (TGF-β) and platelet growth factor BB (PDGF-BB) in SSc-ECs when co-cultured with both HC- and SSc-MSCs; iii. an increase of TGF-β, PDGF-R, alpha smooth muscle actin (α-SMA) and collagen 1 (Col1) in both HC- and SSc-MSCs when co-cultured with SSc-ECs.

Conclusion

We showed that during SSc, the ECs-MSCs crosstalk resulted in an altered expression of different molecules involved in the angiogenic processes, and mainly SSc-ECs seem to modulate the phenotypic switch of perivascular MSCs toward a myofibroblast population, thus supporting the fibrotic process.     

Docosahexaenoic acid induces apoptosis in CYP2E1-containing HepG2 cells by activating the c-Jun N-terminal protein kinase related mitochondrial damage

Docosahexaenoic acid (DHA) causes apoptosis of various cancer cells, but the mechanism of DHA-induced cell death is still unclear. We hypothesized that the early signaling of apoptosis may be important in causing cell death as well as the production of free radical metabolites. DHA caused time- and dose-dependent cell death in human HepG2 hepatoma cells transduced with CYP2E1 (E47) but not in C34 (without CYP2E1), suggesting an important role of CYP2E1 in the DHA-mediated damage. DHA increased the c-Jun N-terminal protein kinase (JNK) activity until 8 h without activating other mitogen-activated protein kinases. The contents of proapoptotic Bad and FasL at 4 h and cytochrome c and caspase 3 activity at 8 h were increased and accompanied by the JNK activation in a successive manner. In contrast, Bax and Bcl-2 were not changed. Levels of lipid peroxides (LPOs) were elevated three- and fivefold at 8 and 24 h, respectively, in DHA-induced E47 cells. However, pretreatment with chlormethiazole (CMZ), a specific inhibitor of CYP2E1, significantly reduced the levels of LPO, CYP2E1, JNK activity and the rate of cell death. In addition, pretreatment with quercetin (one as a JNK inhibitor and one as an antioxidant) significantly reduced the cell death rate and JNK and SEK-1 activities. Our results indicated that DHA-mediated apoptosis in E47 cells was induced through the activation of the JNK-related cell death pathway, which may be involved in the production of LPO or reactive oxygen species during the CYP2E1 catalytic cycle, followed by mitochondrial injury and apoptosis.     

Role of calcium and calcium-activated proteases in CYP2E1-dependent toxicity in HEPG2 cells.

The objective of this work was to investigate whether CYP2E1- and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca2+ and activation of Ca2+-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 microm) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 microm) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca2+ were inhibited by omission of Ca2+ from the extracellular medium, and toxicity was restored by reincorporation of Ca2+. An inhibitor of Ca2+ release from intracellular stores did not prevent the toxicity or the increase in Ca2+, reflecting a role for the influx of extracellular Ca2+ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca2+ in E47 cells exposed to iron-AA were inhibited by alpha-tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40-fold). The toxicity in E47 cells mirrored calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca2+ and activation of Ca2+-dependent proteases.